【作者】 沈报春；

【导师】 徐秀珠；

【作者基本信息】 浙江大学 ， 分析化学， 2007， 博士

【摘要】 本论文是关于大环抗生素及其衍生物手性固定相的研制及药物对映体分离的研究，主要包括大环抗生素手性柱的制备、手性化合物对映体的分离及影响对映体分离各要素的考察与手性识别机理的探讨。论文的第一章为引言，简要介绍了对映体分离的意义和常用方法。论文第二章对大环抗生素手性固定相、多糖衍生物手性固定相、Pirkle型手性固定相的研究及进展进行简要的评述。论文第三章用替考拉宁手性固定相（TE CSP）分别与间甲基苯基异氰酸酯和对氯苯基异氰酸酯反应得到了两种新型的手性固定相—间甲基苯基异氰酸酯替考拉宁手性固定相（MP-TE CSP）和对氯苯基异氰酸酯替考拉宁手性固定相（PCI-TE CSP）。在反相及极性流动相中对替考拉宁及其衍生物CSP的对映体分离能力进行了评价和比较。在反相流动相中，考察了有机添加剂的种类和浓度，缓冲液的pH值等条件对7种氨基酸化合物和3种非氨基酸化合物在三种CSP上对映体分离的影响，计算得出了溶质在三种CSP上的手性选择性自由能差值，初步探讨了这些溶质在三种CSP上的手性识别机理。氨基酸在三种CSP上的保留按以下顺序递减：TE＞PCl-TE＞MP-TE，手性选择性和分离度的顺序则是：MP-TE＞PCl-TE＞TE。10种溶质中，只有去甲羟安定在TE上的分离效果比在MP-TE上稍好，其余溶质对映体都是在两种衍生物CSP上的分离效果比在TE上更好。在极性流动相中，用三种CSP对6种氨基醇类化合物进行了对映体分离研究。固定酸／碱的比例、固定酸（或碱）改变碱（或酸）的浓度，考察流动相中酸碱添加剂的改变对氨基醇类化合物在这三种CSP上对映体分离的影响。氨基醇类化合物在三种CSP上的保留顺序为：PCl-TE＞MP-TE＞TE。MP-TE是三者中对氨基醇类化合物分离效果最差的CSP；PCl-TE CSP对6种氨基醇化合物中的4种（67％）分离效果更好，2种（33％）在TE CSP上表现更佳。论文第四章用三种本实验室自制的手性固定相，替考拉宁（TE），苯基异氰酸酯替考拉宁（Phe-TE）和3，5-二甲基苯基异氰酸酯替考拉宁（DMP-TE CSP）在反相及极性有机溶剂两种流动相体系中对19种化合物进行了对映体分离研究。Phe-TE和DMP-TE CSP是通过衍生化试剂，苯基异氰酸酯、3，5-二甲基苯基异氰酸酯，分别和TE CSP反应而得。与TE相比，衍生物CSP减少了羟基／胺基增加了芳环。在不同的流动相中，不同的作用力对溶质的保留和手性识别起了不同的作用。在反相模式中，溶质与CSP之间的静电作用、疏水作用、氢键作用和空间位阻等共同影响着手性识别。三种CSP对氨基酸的手性分离能力顺序为：DMP-TE＞Phe-TE＞TE。通过计算各CSP间的手性选择性自由能差值发现，用TE与Phe-TE、DMP-TE CSP分别比较时，11种化合物中的55％在Phe-TE CSP上分离效果更好；64％的化合物在DMP-TE CSP上获得更好分离。流动相pH值对衍生物CSP的影响较TE稍大。在极性流动相模式中，溶质与CSP之间的π-π作用，氢键作用和空间位阻共同参与了手性识别。在极性模式下，与Phe-TE和DMP-TE CSP相比，TE是分离β-受体阻滞剂的最佳选择。从机理学观点看，衍生物基团通过4种方式参与手性识别。论文第五章用万古霉素手性固定相（Van CSP）分别和间甲基苯基异氰酸酯和对氯苯基异氰酸酯反应得到两种新型手性固定相：间甲基苯基异氰酸酯万古霉素CSP（MP-Van CSP）和对氯苯基异氰酸酯万古霉素CSP（PCl-Van CSP）。在极性和正相模式中，用这三种CSP分别对三种溶质进行了对映体分离研究。在极性流动相中，考察了甲醇流动相中酸碱添加剂的浓度和比例对手性分离的影响。在Van和MP-Van CSP上，弱碱性药物拉米呋定及其中间体都是在MP-Van CSP上保留更强而在Van CSP上分离效果更好；近中性药物酞胺哌啶酮在MP-Van CSP上保留更强选择性也更大，且流动相中的酸碱浓度对它的影响较小。在正相模式下，对三种轴手性化合物在三种CSP上进行了对映体分离研究。改变流动相中醇类添加剂的种类和浓度，探讨了三种CSP的手性识别机理。联二萘酚只在MP-Van CSP上得到部分分离；联二萘胺在MP-Van CSP上保留更强而在Van CSP上分离效果更好；联二萘酚胺在MP-Van CSP上保留更强选择性也更大。由于溶质在PCl-Van CSP上的理论塔板数与在另外两种CSP上的理论塔板数值相差较大，溶质在该CSP上的对映体分离没有和前两个CSP进行比较。论文第六章用手性柱和非手性柱对一个自己合成的扁桃酸衍生物，2-苯基-2-四氢吡喃氧基醋酸甲酯进行了光学异构体分离研究。由于其分子中含有2个-手性中心，溶质有4个光学异构体。用（S，S）-Whelk-O1手性柱进行分离时，得到了4个完全分离的色谱峰。考察流动相添加剂对溶质光学异构体分离的影响时发现，当用甲基叔丁基醚（TBME）作为有机添加剂时，溶质得到了最好的分离效果。但是流动相中的TBME却影响了溶质的保留行为，光学异构体的保留因子在含TBME的流动相中发生了漂移。而且TBME的浓度还影响了保留因子漂移的程度，溶质在含5％（v／v）TBME的流动相中的保留因子漂移比在含8％（v／v）和10％（v／v）TBME的流动相中都要大。当流动相添加剂换成异丙醇时，保留因子漂移现象消失。溶质在CDMPC手性柱上的光学异构体分离效果并不理想，只获得了3个色谱峰。在三种非手性柱（C18，硅胶柱和气相色谱HP-5柱）上，溶质分别获得了2个峰。论文第七章在O，O′-二（3，5-二甲基苯甲酰）Ⅳ，Ⅳ-二烯丙基酒石酸二酰胺（CHI-DMB）及（R，R）-3，5-二硝基苯甲酰基-1，2-二苯基乙烷-1，2-二胺（（R，R）-DNB-DPEDA）手性柱上对喜树碱合成中间体—-2-[N-对甲苯磺酰基-（R）-脯氨酰氧基]-2-[6-氰基-（1，1-亚乙二氧基）-5-酮-1，2，3，9-四氢中氮茚-7-基]-丁酸乙酯进行了光学异构体分离研究。考察了流动相中极性醇类添加剂的种类及浓度对分离的影响，并比较了溶质在这两种手性柱上的手性识别机理。在这两种手性固定相上，溶质与手性固定相之间的吸引作用都是产生手性识别的关键。从溶质与固定相的空间结构看，在CHI-DMB手性柱上，π-π堆积作用及偶极偶极作用是关键的；而在（R，R）-DNB-DPEDA手性柱上，π-π堆积作用，偶极偶极作用及氢键作用对分离有利。此外，空间位阻在喜树碱合成中间体光学异构体分离中也起了一定的作用。根据溶质和固定相的空间结构，推导出两个光学异构体的流出顺序，并通过相应的光学异构体得到验证。论文第八章在两大类五种手性固定相上对两种新型有机膦化合物，[（2-氯-苯胺）-（2-羟基-苯基）]-膦酸二乙酯（化合物1）和[（2-氯-苯胺）-（4-氟-苯胺）-甲基]-膦酸二乙酯（化合物2）进行了对映体分离研究。分别考察了在正己烷流动相中醇类添加剂的种类及浓度对手性分离的影响。同时，通过研究溶质的立体结构因素对手性分离的影响，探讨了这两种有机膦化合物在不同手性柱上的手性识别机理。化合物1在（S，S）-Whelk-O1，CHI-DMB，CDMPC和纤维素三苯基氨基甲酸酯（CTPC）手性柱上得到了分离；化合物2在（R，R）-DNB-DPEDA和CDMPC上获得分离。在三种“刷型”CSP上，溶质和CSP之间的吸引作用对手性识别是非常必要的。在（S，S）-Whelk-O1和CHI-DMB手性柱上，溶质和CSP之间的氢键作用对手性识别较为重要；在（R，R）-DNB-DPEDA手性柱上，溶质和CSP之间的偶极偶极作用和π-π作用对手性识别有利。在（S，S）-Whelk-O1和（R，R）-DNB-DPEDA上，建立了溶质流出顺序和绝对构型的关系图。在纤维素衍生物CSP上，流动相中的酸添加剂是必需的。溶质和CSP之间的空间适应性对手性识别较为关键。此外，化合物1和CTPC之间的氢键作用对手性识别较重要，但是它和CDMPC之间的氢键作用对手性识别影响较小。论文第九章在CDMPC和万古霉素手性固定相上分别分离了稳杀得、千金和禾草灵三种芳氧苯氧丙酸类除草剂的对映异构体。在这两种手性固定相上分别考察了流动相组成和溶质的结构对手性分离的影响。同时探讨了三种溶质在两种CSP上的手性识别机理。三种芳氧苯氧丙酸类除草剂中，千金在CDMPC上保留最强而禾草灵分离效果最好。万古霉素手性固定相对三种溶质的分离效果明显不如CDMPC。本章使用的方法为寻找高效低毒的芳氧苯氧丙酸类除草剂提供了技术支持。论文第十章在正相模式下，用直链淀粉衍生物手性固定相Chiralpak AD柱对二茂铁衍生物—3-（1-N，N-二甲基）乙基二茂铁进行了对映体分离研究，考察了醇类添加剂种类和浓度，碱的浓度以及温度对对映体分离的影响。乙醇作为醇类添加剂对AD柱手性空腔立体环境的改变最有利于溶质在AD柱上的对映体分离。流动相中碱的加入改善了峰形，在一定程度上提高了分离度。热力学研究表明在所研究的温度范围内（15-35℃），固定相和溶质之间的相互作用没有发生改变。此外，△△H⁰和△△S⁰均为负值，说明该系列化合物的拆分为焓驱动过程，温度升高对分离不利。同时，计算出了对映体分离转折温度T_iso。更多还原

【Abstract】 This dissertation focuses on the enantioseparation using macrocyclic antibioticschiral stationary phases （CSPs） by high performance liquid chromatography （HPLC）,including preparation of macrocyclic glycopeptide and its phenyl isocyanatederivatives, enantioseparation of chiral compounds, study of several factorsconcerning chiral separation and discussion of chiral recognition mechanism.In Chapter 1, the importance and methods of chiral separation is brieflyintroduced.In Chapter 2, the literatures of three kinds of CSPs, macrocyclic CSPs,polysaccharide derivative CSPs and Pirkle type CSPs are briefly reviewed.In Chapter 3, two new CSPs, m-methylphenyl isocyanate teicoplanin （MP-TE）and p-chlorophenyl isocyanate teicoplanin （PCl-TE） are synthesized from themacrocyclic glycopeptide teicoptanin CSP（TE）, using derivative agents,m-methylphenyl isocyanate or p-chlorophenyl isocyanate. Evaluation and comparisonof these three TE-based CSPs are processed with 16 recemates under RP （reversedphase） and PO （polar organic） mode, respectively. The chromatographic results aregiven as the retention, selectivity, resolution factors and the enantioselective freeenergy difference corresponding to the separation of the two enantiomers. The effectof pH, organic modifier type and amount are discussed, and the stereoselectivity forthree TE-based CSPs are compared. The retention forα-amino acids on these threeCSPs follow the direction:TE＞PCl-TE＞MP-TE, while chiral selectivity andresolution follow the direction: MP-TE＞PCl-TE＞TE. Compare TE with eachderivative CSP, 1 compound （10%） （oxazapam） gets better separation on TE and 9（90%） gets better on MP-TE CSP; all compounds get better separation on PCl-TECSP, under RP mode. 6 amino alcohols are enantioseparated on three TE-based CSPsunder PO mode. The influence of the amount and the ratio of the acid/alkali on theenantioseparation are investigated. The retentions for amino alcohols of these threeCSPs follow the direction:PC1-TE＞MP-TE＞TE and the selectivities of these threeCSPs show great differences. MP-TE CSP is the most unfavorable CSP for enantiosepation ofβ-blockers using PO mobile phase. Compare TE with PC1-TE CSP,4β-blockers（67%） get better enantiomeric separation on PCl-TE and 2（33%） getbetter on TE CSP, under PO mode.In Chapter 4, HPLC enantiomeric separations of 19 racemic analytes, 8 aminoacids and 11 non amino acids, are evaluated using chiral stationary phases （CSPs）based on the macrocyclic glycopeptides teicoplanin （TE）, phenylisocyanateteicoplanin （Phe-TE）, and 3, 5-dimethylphenylisocyanate teicoplanin （DMP-TE） intwo different mobile phase modes, i.e., the RP mode and the PO mode. The Phe-TEand the DMP-TE CSPs are synthesized from the TE using derivative agents, phenylisocyanate or 3, 5-dimethylphenyl isocyanate. The chromatographic results are givenas the retention, selectivity, and resolution factors and the enantioselective free energydifference corresponding to the. separation of the two enantiomers. The effect of pH,organic modifier type and amount are discussed, and the stereoselectivity for threeTE-based CSPs are compared. The chiral selectivity forα-amino acids of these threeCSPs follows the direction: DMP-TE＞Phe-TE＞TE. Compare TE with each derivativeCSP, 6 compounds（55%） get better separation on Phe-TE and 5（45%） better on TECSP; 7 compounds（64%） get better separation on DMP-TE and 4（36%） on TE, underRP mode. The TE is the favorable CSP for enantioseparation theβ-blockers using POmobile phase. Comparison of the enantiomeric separations using self-made TE,Phe-TE, and DMP-TE are conducted in order to gain a better understanding of thechiral recognition mechanism of the macrocyclic glycopeptide antibiotics CSRIn Chapter 5, two new CSPs, m-methylphenyl isocyanate vancomycin （MP-Van）,and p-chlorophenyl isocyanate vancomycin （PC1-Van） are synthesized from themacrocyclic glycopeptide vancomycin CSP （Van）, usingi derivative agents,m-methylphenyl isocyanate or p-chlorophenyl isocyanate. Evaluation and comparisonof these three Van-based CSPs are processed with 3 recemates under PO and NP（Normal phase） mode, respectively. In PO mode, the influence of the ratio and theamount of acid/alkali on the enantioseparion is investigated and the chiral recognitionmechanism is discussed. The weak alkali pharmaceutical lamivudine and itsintermediate retain longer on MP-Van while enantioseparate better on Van CSP. The near-neutral pharmaceutical thalidomide gets greater retention and selectivity both onMP-Van CSR Furthermore, the acid and/or alkali give little influence on itsenantiomeric separation. Under NP mode, three axis chiral analytes are separated onthree Van-based CSPs. The influence of the type and the amount of alcoholicadditives and solute structure on the chiral separation is studied. 2, 2’-dihydroxy-1,1’-binaphthyl can be partly enantioseparated on MP-Van CSR 2, 2’-diamino-1,1’-binaphthyl gets longer retention on MP-Van CSP and better selectivity on Van CSR2-amino-2’-hydroxy-1, 1’-binaphthyl gets greater retention and selectivity both onMP-Van CSP.In Chapter 6, conditions for separation of isomers of a self-prepared mandelicacid derivative, methyl 2-phenyl-2-（tetrahydropyranyloxy） acetate （the analyte）were studied. Because of the presence of two chiral carbons, the analyte consists offour stereoisomers stable at ambient temperature with 2SS-, 2RS-, 2SR-, and2RR-configurations （2SS, the isomer with 2S, 1S-configuration, etc.）. Chiral HPLC ofthe analyte results in four peaks, using the （S, S）-Whelk-O1 with the mobile phaseconsisting of hexane and the t-butyl methyl ether （TBME）, suggesting that thecolumn is capable of resolving the analyte sufficiently. It is found that TBMEpresence dramatically changed the retention activity of the isomers though it producesthe best enantioseparation on （S, S）-Whelk-O1 chiral column. The amount of TBMEin the mobile phase influences the degree of retention change. The 5% （v/v） TBMEgives the biggest shift compares with concentration 10% （v/v） and 8% （v/v）. Underthe same condition, 2-propanol does not produce the same results. The chiralseparation is also tried on cellulose tris （3, 5-dimethylphenylcarbamate） （CDMPC）and gets 3 peaks to four isomers indicating some but not full enantiomer resolution.Achiral HPLC of the analyte results in two well-resolved peaks when analyzed onthe C18 column under RP mode and on the #-porasil column using normal phasemobile phase, respectively. The two peaks correspond to the two enantiomer pairs,2SS/2RR and 2RS/2SR, but with, at this stage, unknown elution order. The achiralseparation is also got on HP-5 column by GC, MS confirmes the structure. The chiral and achiral separations of diastereoisomers 2R and 2S of the analyte areexperimented simultaneously （2R, the diastereoisomer with 2R fixed, etc.）.In Chapter 7, the separation of isomers of camptothecin synthetic intermediate,2-[-p-tosyl-（R）-prolinoyl]-2-[6-cyanoyl-（1,1-ethylenedioxy）-5-one-1,2,3,9-quarthydro-indolizine-yl] ethyl butyrate, on O,O’-bis （3,5-dimethylbenzoyl）-N,N’-diallyl-L-tartardiamicle（CHI-DMB） and（R, R）-3,5-dinitrobenzoyl-1,2- diphenylethane-1,2-diamine（（R, R）-DNB-DPEDA） chiral columns is got. The influence of the alcoholic modifiersin mobile phase, including kinds and the concentration for the separation is studied.We also investigate the separation mechanism of the analyte on the two chiralcolumns and find that the attractive interactions between the analyte and the CSP playthe predominant role on both CSPs. Consulted from the interactions between theCSP and the analyte, on CHI-DMB’ chiral column,π-πstacking, dipole-dipoleinteractions react whileπ-πstacking, dipole-dipole interaction and the hydrogenbonding work on （R, R）-DNB-DPEDA chiral column. Besides these, the steric bulk isalso important for the separation of the isomers of camptothecin syntheticintermediate. A rational correlation is found between elution order and absoluteconfiguration of the analyte. The elution order is confirmed by corresponding isomer.In Chapter 8, direct enantiomeric resolution of two synthesized organicphosphonate derivatives [（2-Chloro-phenylamino）-（2-hydroxy-phenyl）-methyl]phosphonic acid diethyl ester（compound 1）and [（2-Chloro-phenyl）-（4-fluoro-phenyl-amino）-methyl] phosphonic acid diethyl ester（compound 2） have been achieved,using hexane as the mobile phase with various alcohols as modifiers. The influence ofthe mobile phase composition and solute structure on the chiral separation is studied.It is found that compound 1 is achieved well separation on （S,S）-Whelk-O1,CHI-DMB, cellulose tris（3,5-dimethylphenylcarbamate）（CDMPC） and cellulosetrisphenylcarbamate （CTPC） chiral stationary phases while compound 2 could beseparated on （R, R）-DNB- DPEDA and CDMPC chiral stationary phases. The chiralrecognition mechanism of （S, S）-Whelk-O1, （R, R）-DNB-DPEDA and cellulosederivative CSPs is explored. On （S, S）-Whelk-O1 and CHI-DMB CSP, hydrogen-bonding interactions play an important role in chiral recognition. On （R,R）-DNB-DPEDA-CSP, the dipole-dipole interactions andπ-πstacking are importantto chiral discrimination. On （S, S）-Whelk-O1 CSP and （R, R）-DNB-DPEDA CSP, acorrelation is concluded between elution order and absolute configuration of theanalytes. Interestingly, on cellulose derivative CSPs, acidic additive is necessary forenantioseparation of 1 and 2. On cellulose CSPs, the inclusion and fitness of soluteshape in the chiral cavity significantly contributes to the enantioseparation of solutes.Furthermore, as for the enantioseparation 1, the hydrogen-bonding interactions playan important role on CTPC, but play a minor role on CDMPC.In Chapter 9, three high efficiency aryloxyphenoxypropionic acid herbicides,fluazifop, cyhalofop-butyl and diclofop, are enantioseparated on CDMPCsuccessfully. The influence of the mobile phase composition and solute structure onthe chiral separation is studied. Among three aryloxyphenoxypropionic acidherbicides, the cyhalofop-butyl retains longest and diclofop gets the bestenantioseparation indicating that the dipole-dipole interaction orπ-πstacking whichproduced by CN group of the cyhalofop-butyl might benefit to retention but do notcontribute to enantioselectivity. The results also show that the hydrogen-bondinginteractions between the fluazifop and CDMPC play an important role in chiralrecognition. As for diclofop and cyhalofop-butyl, the inclusion and fitness of soluteshape in the chiral cavity contribute to the chiral discrimination dramatically. Inconclusion, the structure of solutes plays a key role in the chiral recognition. Threearyloxyphenoxypropionic acid herbicides are also experimented on vancomycin CSPunder normal phase but the results are not as good as that of on CDMPC. The methodbrings out a technical support for searching for high efficiency and low poisonaryloxyphenoxypropionic acid herbicides.In Chapter 10, the enantiomeric separation of a ferrocene derivative, 3-（1-N,N-dimethyl）-ethyl ferrocene, is got using an amylose tris（3,5-dimethylphenyl-carbamate）（Chiralpak AD） chiral column, under normal phase mode. The influence ofthe mobile phase composition on the chiral separation is studied. Ethanol is the bestalcoholic additive because the twisting of the glucose unit caused by it just fit in with the shape of the solute. The alkali in the mobile phase makes the peak shape betterbut does not good for selectivity. The thermodynamic results show that the interactionbetween the solute and the CSP do not change in the research temperature range（from 15 to 35℃）. Furthermore, the values of△△H°. and△△S°are both negative,which indicate this is an enthalpy-driven separation. The increase of the columntemperature is not good for the enantioseparaion. The enantioseparation transitiontemperature T_iso is 601K.更多还原